Sensitive and label-free T4 polynucleotide kinase/phosphatase detection based on poly(thymine)-templated copper nanoparticles coupled with nicking enzyme-assisted signal amplification

Dephosphorylation of DNA by DNA 30-phosphatases plays significant regulatory roles in DNA damage repair, replication, and recombination. Here, we present a novel, highly sensitive and label-free fluorescent assay for T4 polynucleotide kinase/phosphatase (T4 PNKP) activity and its inhibitors on the basis of nicking enzyme-assisted signal amplification and poly(thymine)-templated fluorescent copper nanoparticles (CuNPs) as a fluorescent indicator. It was designed such that an A-rich hairpin primer with a 30-phosphoryl end can serve as the substrate for T4 PNKP. Once the phosphorylated hairpin primer was hydrolyzed by T4 PNKP, the resulting hairpin primer with a 30-hydroxyl end was immediately elongated to form a long double-strand product using DNA polymerase as well as a double-stranded recognition site for nicking enzyme Nb. BbvCI. With the help of nicking enzyme Nb. BbvCI, the double-strand product could be cleaved to release a single-stranded poly-(thymine) (poly T) DNA, which could be used as a template for the formation of fluorescent CuNPs. Subsequently, the remaining probe could be reacted as the primer to trigger the next cycle of polymerization and scission process, thus significantly improving the sensitivity of the sensor. Furthermore, the application of the proposed strategy for screening T4 PNKP inhibitors also demonstrated satisfactory results. With the advantages mentioned above, this simple, cost-effective, and highly sensitive strategy has potential for the study of DNA damage repair mechanisms.